Examination of the effects of GGA on levels of antioxidant and oxidant products.

<p>(A) The activity of SOD was determined at 500 nm and the values are expressed as U/mg protein. (B) MDA levels were measured at 532 nm and the results are represented as nmol/mg protein. (C) GSH activity was detected at 420 nm and the data are expressed as mg/g protein. Three independent experiments were performed; * p<0.05 and ** p<0.01; ns indicates no significant difference.</p

Effect of GGA on the mitochondria-mediated apoptotic pathway.

<p>(A) Western blot analysis to examine the protein levels of Bcl-2, and cytochrome c in the cytosol and mitochondria in cardiomyocytes. The protein expression of Bcl-2 (B), cytochrome c in the cytosol (C), and mitochondria (D) was analyzed using Image-Pro Plus 6.0 software and normalized to GAPDH, α-tubulin, and VDAC, respectively. (E) Effect of GGA on caspase-3 activity. A total of 20 mg protein lysate of cardiomyocytes was examined by incubation with Ac-DEVD-MCA for 5 min at 37°C. The release of 7-amino-4-methylcoumarin was measured using a spectrofluorometer. * p<0.05 and ** p<0.01.</p

Effects of TMP on MAPKs pathway.

<p>(A) The proteins ERK, p-ERK, p38, p-p38, JNK and p-JNK were determined using western blotting with corresponding antibodies. β-actin was used as an internal control. (B, C, D) Data are expressed as mean±SD; #<i>P</i><0.05 compared with control; *P<0.05 compared with OGD. (E) OGD-induced up-regulation of Cx32 expression was inhibited by both the ERK inhibitor U0126 (5 µM) and the p38 inhibitor SB203580 (5 µM), but not by the JNK inhibitor SP600125 (5 µM). The Cx32 protein was determined using western blotting. β-actin was used as a loading control. (F) Cx32 mRNA expression was detected by qRT-PCR. All data are means±SD (#<i>P</i><0.05 vs. control; *<i>P</i><0.05 vs. OGD/R). (G) MTT cell viability of normal control, OGD and U0126 (5 µM), SB203580 (5 µM) and SP600125 (5 µM) treatment groups (9 wells per group). Data are expressed as mean±SD; #<i>P</i><0.05 vs. control, *<i>P</i><0.05 vs. OGD. (H) LDH release assay normal control, OGD and U0126 (5 µM), SB203580 (5 µM) and SP600125 (5 µM) treatment groups. Data are expressed as mean±SD; #<i>P</i><0.05 vs. control, *<i>P</i><0.05 vs. OGD (I) The proteins Bcl-2 and Bax were determined using western blotting with corresponding antibodies. β-actin was used as an internal control. All experiments were repeated at least three times.</p

Effect of GGA on Hsp70 expression profiles.

<p>Hsp70 mRNA expression (A) and protein expression (B) in cardiomyocytes were analyzed by qRT-PCR and Western blot analysis, respectively. (C) The protein expression was analyzed using Image-Pro Plus 6.0 software and normalized to GAPDH. RT/Vehicle, mice pretreated with vehicle and kept at room temperature acted as controls; RT/GGA, mice pretreated with GGA and kept at room temperature; HHS/Vehicle, mice pretreated with vehicle and treated with HHS; HHS/GGA, mice pretreated with GGA and treated with HHS. *p<0.05 and **p<0.01.</p

Analysis of apoptotic cells by TUNEL staining.

<p>Mean numbers of TUNEL-positive cells in a field of 500 × 500 μm. ** p<0.01.</p

Effect of TMP on OGD-induced apoptosis in cultured hippocampal neurons.

<p>(A) Hippocampal neurons apoptosis was detected via PI staining and the Annexin V method. Data acquisition was conducted by collecting 20,000 cells per tube and the numbers of viable and dead cells were determined for each experimental condition. TMP (10 and 20 µM) and caspase inhibitor (zVAD-fmk 50 uM) were applied from OGD to the end of experiments. Controls and OGD group received the same amount of DMSO. The camptothecin group, neurons were exposure to 10 µM camptothecin used to experiment. (B) Columns show the mean of data obtained from three independent experiments. Bar mean SD. n = 3, * <i>P</i><0.05 vs. control, ^#<i>P</i><0.05 vs OGD.</p

TMP treatment down-regulates the expression of Cx32 in hippocampal neurons.

<p>(A) The proteins Cx32 was determined using western blotting. β-actin was used as a loading control. (B) Cx32 mRNA expression is detected by qRT-PCR. All experiments were repeated at least three times. All data are means±SD. (n = 3) (#<i>P</i><0.05 vs. control; *<i>P</i><0.05 vs. OGD). (C) Uptake of the connexin permeable dye lucifer yellow (LY) was measured to confirm that Cx32 is activated during OGD and blocked by TMP. Values represent the counts of 9 wells per group±SD. (n = 3) (#<i>P</i><0.05 vs. control; *<i>P</i><0.05 vs. OGD).</p

Effect of TMP on neurons viability.

<p>(A) MTT cell viability of normal control, OGD and TMP (10 and 20 µM) treatment groups (9 wells per group). Data are expressed as mean±SD; n = 9; # <i>P</i><0.05 vs. control, *<i>P</i><0.05 vs. OGD. (B) LDH release assay of normal control, OGD and TMP (10 and 20 µM) treatment groups. After OGD, increased LDH release was significantly attenuated by TMP. Data are expressed as mean±SD; n = 3, ^#<i>P</i><0.05 vs. control, *<i>P</i><0.05 vs. OGD.</p

Effects of Cx32 overexpression on neuron viability.

<p>(A) Western blot analysis of the protein levels of Cx32 in neuron, β-actin was used as a loading control. (B) Cx32 mRNA expression levels expression is detected by RT-PCR. (C) After OGD, MTT cell viability assay of normal control, Vector and Cx32 overexpression groups. (D) LDH release of normal control, Vector and Cx32 overexpression groups. All experiments were repeated at least three times. Data are expressed as mean±SD; *<i>P</i><0.05 vs. control.</p

Inhibition of Cx32 expression and OGD -induced injury by siRNA-Cx32 in cultured hippocampal neurons.

<p>(A) Western blot analysis of Cx32 expression at 48 h after transfected with siCx32 and siMock. β-actin was used as a loading control. (B) Cx32 mRNA expression was detected by qRT-PCR at 48 h after transfected with siCx32 and siMock. All data are means±SD (*<i>P</i><0.05 vs. control). (C) The photo images show that treatment of hippocampal neurons with siCx32 attenuated OGD-induced increase in LY-positive cells. (D) After OGD, MTT cell viability assay of normal control, siMock and siCx32 groups (9 wells per group). Data are expressed as mean±SD; *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. OGD. (E) LDH release assay of normal control, OGD and TMP (10 and 20 µM) treatment groups. Data are expressed as mean±SD; *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. OGD. (F) Hippocampal neurons apoptosis was detected via PI staining and the Annexin V method. Data acquisition was conducted by collecting 20,000 cells per tube and the numbers of viable and dead cells were determined for each experimental condition. Columns show the mean of data obtained from three independent experiments. Bar mean SD. * <i>P</i><0.05 vs. control, ^#<i>P</i><0.05 vs OGD. (G) The proteins Bcl-2 and Bax were determined using western blotting with corresponding antibodies. β-actin was used as an internal control. All experiments were repeated at least three times.</p